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Comparison of Methods to Identify Pathogens and Associated Virulence Functional Genes in Biosolids from Two Different Wastewater Treatment Facilities in Canada.

Yergeau E, Masson L, Elias M, Xiang S, Madey E, Huang H, Brooks B, Beaudette LA - PLoS ONE (2016)

Bottom Line: Various regulatory bodies typically employ indicator organisms (fecal coliforms, E. coli and Salmonella) to assess the adequacy and efficiency of the wastewater treatment process in reducing pathogen loads in the final product.Our results show that an anaerobic digestion WWTP was unsuccessful at reducing the live indicator organism load (coliforms, generic E. coli and Salmonella) below acceptable regulatory criteria, while biosolids from a dewatering/pelletization WWTP met these criteria.Clostridium DNA increased significantly following anaerobic digestion treatments.

View Article: PubMed Central - PubMed

Affiliation: National Research Council Canada, Energy Mining and Environment, Montreal, Qc, Canada.

ABSTRACT
The use of treated municipal wastewater residues (biosolids) as fertilizers is an attractive, inexpensive option for growers and farmers. Various regulatory bodies typically employ indicator organisms (fecal coliforms, E. coli and Salmonella) to assess the adequacy and efficiency of the wastewater treatment process in reducing pathogen loads in the final product. Molecular detection approaches can offer some advantages over culture-based methods as they can simultaneously detect a wider microbial species range, including non-cultivable microorganisms. However, they cannot directly assess the viability of the pathogens. Here, we used bacterial enumeration methods together with molecular methods including qPCR, 16S rRNA and cpn60 gene amplicon sequencing and shotgun metagenomic sequencing to compare pre- and post-treatment biosolids from two Canadian wastewater treatment plants (WWTPs). Our results show that an anaerobic digestion WWTP was unsuccessful at reducing the live indicator organism load (coliforms, generic E. coli and Salmonella) below acceptable regulatory criteria, while biosolids from a dewatering/pelletization WWTP met these criteria. DNA from other pathogens was detected by the molecular methods, but these species were considered less abundant. Clostridium DNA increased significantly following anaerobic digestion treatments. In addition to pathogen DNA, genes related to virulence and antibiotic resistance were identified in treated biosolids. Shotgun metagenomics revealed the widest range of pathogen DNA and, among the approaches used here, was the only approach that could access functional gene information in treated biosolids. Overall, our results highlight the potential usefulness of amplicon sequencing and shotgun metagenomics as complementary screening methods that could be used in parallel with culture-based methods, although more detailed comparisons across a wider range of sites would be needed.

No MeSH data available.


Bacterial phylum-level community composition (a) and genus-level principal coordinate analysis (PCoA) ordination (b) for 16S rRNA gene sequencing of the V1–V3 and V3–V5 regions, cpn60 gene sequencing and shotgun metagenomic sequencing for pre- and post-treatment biosolid sampled at Plant A on August 4, 2009.Solid symbols: pre-treatment, empty symbols: post-treatment.
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pone.0153554.g002: Bacterial phylum-level community composition (a) and genus-level principal coordinate analysis (PCoA) ordination (b) for 16S rRNA gene sequencing of the V1–V3 and V3–V5 regions, cpn60 gene sequencing and shotgun metagenomic sequencing for pre- and post-treatment biosolid sampled at Plant A on August 4, 2009.Solid symbols: pre-treatment, empty symbols: post-treatment.

Mentions: One pre/post sample pair from Plant A (August 4, 2009) and another pair from Plant C (May 12, 2009) were selected for amplicon sequencing (16S and cpn60). For both plants, large changes were observed at the phylum/class level following treatment independent of the primer pair used (Fig 1a). For Plant A, Proteobacteria dominated the microbial community before treatment and was replaced by Firmicutes, Bacteroidetes and Chloroflexi following treatment (Fig 1a). For Plant C, the shifts were less drastic, with decreases in the relative abundance of Proteobacteria and increases in Actinobacteria, Bacteroidetes, and Firmicutes following treatment (Fig 1a). When looking at lower taxonomical levels using Unifrac analyses, a similar pattern emerged with the anaerobic digestion from Plant A causing stronger shifts in microbial communities (dots more distant) and the dewatering/pelletization from Plant C causing less dramatic shifts (Fig 1b). Similar results were obtained from cpn60 amplicon analyses for Plant A (Fig 2) and Plant C (not shown). Shifts in the dominant genera were also observed for the two plants following treatments using 16S rRNA and cpn60 gene sequencing. For Plant A, the community shifted from an Acidovorax and Novosphingobium dominated community (Table 2) to one dominated by anaerobes and syntrophic bacteria like Syntrophus, Sedimentibacter, Prevotella, Clostridium and Thermovirga (Table 3). For Plant C, the shifts were less evident, with a community that was generally dominated by Paludibacter and Microbacterium both before and after the biosolid treatment (Table 4).


Comparison of Methods to Identify Pathogens and Associated Virulence Functional Genes in Biosolids from Two Different Wastewater Treatment Facilities in Canada.

Yergeau E, Masson L, Elias M, Xiang S, Madey E, Huang H, Brooks B, Beaudette LA - PLoS ONE (2016)

Bacterial phylum-level community composition (a) and genus-level principal coordinate analysis (PCoA) ordination (b) for 16S rRNA gene sequencing of the V1–V3 and V3–V5 regions, cpn60 gene sequencing and shotgun metagenomic sequencing for pre- and post-treatment biosolid sampled at Plant A on August 4, 2009.Solid symbols: pre-treatment, empty symbols: post-treatment.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4835084&req=5

pone.0153554.g002: Bacterial phylum-level community composition (a) and genus-level principal coordinate analysis (PCoA) ordination (b) for 16S rRNA gene sequencing of the V1–V3 and V3–V5 regions, cpn60 gene sequencing and shotgun metagenomic sequencing for pre- and post-treatment biosolid sampled at Plant A on August 4, 2009.Solid symbols: pre-treatment, empty symbols: post-treatment.
Mentions: One pre/post sample pair from Plant A (August 4, 2009) and another pair from Plant C (May 12, 2009) were selected for amplicon sequencing (16S and cpn60). For both plants, large changes were observed at the phylum/class level following treatment independent of the primer pair used (Fig 1a). For Plant A, Proteobacteria dominated the microbial community before treatment and was replaced by Firmicutes, Bacteroidetes and Chloroflexi following treatment (Fig 1a). For Plant C, the shifts were less drastic, with decreases in the relative abundance of Proteobacteria and increases in Actinobacteria, Bacteroidetes, and Firmicutes following treatment (Fig 1a). When looking at lower taxonomical levels using Unifrac analyses, a similar pattern emerged with the anaerobic digestion from Plant A causing stronger shifts in microbial communities (dots more distant) and the dewatering/pelletization from Plant C causing less dramatic shifts (Fig 1b). Similar results were obtained from cpn60 amplicon analyses for Plant A (Fig 2) and Plant C (not shown). Shifts in the dominant genera were also observed for the two plants following treatments using 16S rRNA and cpn60 gene sequencing. For Plant A, the community shifted from an Acidovorax and Novosphingobium dominated community (Table 2) to one dominated by anaerobes and syntrophic bacteria like Syntrophus, Sedimentibacter, Prevotella, Clostridium and Thermovirga (Table 3). For Plant C, the shifts were less evident, with a community that was generally dominated by Paludibacter and Microbacterium both before and after the biosolid treatment (Table 4).

Bottom Line: Various regulatory bodies typically employ indicator organisms (fecal coliforms, E. coli and Salmonella) to assess the adequacy and efficiency of the wastewater treatment process in reducing pathogen loads in the final product.Our results show that an anaerobic digestion WWTP was unsuccessful at reducing the live indicator organism load (coliforms, generic E. coli and Salmonella) below acceptable regulatory criteria, while biosolids from a dewatering/pelletization WWTP met these criteria.Clostridium DNA increased significantly following anaerobic digestion treatments.

View Article: PubMed Central - PubMed

Affiliation: National Research Council Canada, Energy Mining and Environment, Montreal, Qc, Canada.

ABSTRACT
The use of treated municipal wastewater residues (biosolids) as fertilizers is an attractive, inexpensive option for growers and farmers. Various regulatory bodies typically employ indicator organisms (fecal coliforms, E. coli and Salmonella) to assess the adequacy and efficiency of the wastewater treatment process in reducing pathogen loads in the final product. Molecular detection approaches can offer some advantages over culture-based methods as they can simultaneously detect a wider microbial species range, including non-cultivable microorganisms. However, they cannot directly assess the viability of the pathogens. Here, we used bacterial enumeration methods together with molecular methods including qPCR, 16S rRNA and cpn60 gene amplicon sequencing and shotgun metagenomic sequencing to compare pre- and post-treatment biosolids from two Canadian wastewater treatment plants (WWTPs). Our results show that an anaerobic digestion WWTP was unsuccessful at reducing the live indicator organism load (coliforms, generic E. coli and Salmonella) below acceptable regulatory criteria, while biosolids from a dewatering/pelletization WWTP met these criteria. DNA from other pathogens was detected by the molecular methods, but these species were considered less abundant. Clostridium DNA increased significantly following anaerobic digestion treatments. In addition to pathogen DNA, genes related to virulence and antibiotic resistance were identified in treated biosolids. Shotgun metagenomics revealed the widest range of pathogen DNA and, among the approaches used here, was the only approach that could access functional gene information in treated biosolids. Overall, our results highlight the potential usefulness of amplicon sequencing and shotgun metagenomics as complementary screening methods that could be used in parallel with culture-based methods, although more detailed comparisons across a wider range of sites would be needed.

No MeSH data available.